This is the revised version of an application for competitive renewal of grant RO1-GM57691-13. Since its inception (in 1999), we focused on the mechanisms that regulate gap junctions formed by Connexin43 (Cx43). For this application, we take the reverse angle, and look at the ability of Cx43 to be the regulator of other molecular complexes. In its classical definition, the function of Connexin43 (Cx43) is to form gap junctions. While this description is certainly correct, it does not exclude the possibility that Cx43 exerts other actions, separate from that of gap junction pore formation. Here, we propose that a) specific amino acids within the Cx43 carboxyl terminal domain modulate the function of sodium channels, b) this regulation occurs within the confines of the perinexus, that is, the area surrounding a gap junction plaque, and c) Cx43-mediated regulation of cell electrophysiology is necessary for proper cardiac rhythm. Under Aim 1, we will characterize the cardiac electrophysiological profile of mice after loss of Cx43 expression (Cx43-CKO), or after deletion of the last five amino acids of Cx43 (line Cx43D378stop). Both of these mice present with ventricular fibrillation and sudden death. A fundamental difference is that mutation D378stop does not prevent formation of gap junction plaques. We propose that Cx43 is necessary for proper function of other ion channels. In aim 2, we will define the structure of the perinexus in relation to the sodium channel complex, and the importance of preservation of region 378-382 of Cx43 to control the distance between molecules. We implement a unique combination of modern imaging/recording techniques (scanning ion conductance microscopy, SICM-guided patch clamp, tomographic electron microscopy, direct stochastic reconstruction microscopy, proximity ligation assays), and novel animal models, to address new hypotheses that directly impact our understanding of the molecular mechanisms of cardiac arrhythmias. Overall, our experiments challenge the prevailing concept that, in a structurally normal heart, Cx43-dependent arrhythmias are only consequent to the loss of gap junction channels between cells.